The subject matter disclosed herein relates to acquisition and analysis of images of biological samples. More particularly, the disclosed subject matter relates to the calibration of microscopes used in such image acquisition protocols.
Certain types of molecular pathology examinations utilize a multiplexing workflow for molecular pathology imaging. When generating images using such a multiplexing workflow, a single slice of tissue (i.e., a single sample) may be used. The multiplexing workflow allows images of the tissue sample acquired over multiple rounds of imaging to be layered, with each round of imaging being directed to a different set of biomarkers applied to the sample. Through the combination of biomarkers acquired over multiple rounds of imaging, a comprehensive view of tissue composition may be attained for the sample.
By way of example, in one such approach the tissue sample is repeatedly stained and bleached during a given imaging protocol. A round of image acquisition may be performed using an automated fluorescence microscope for background acquisition or after each stain or bleach cycle. For example, after an initial stain application a set of images may be acquired, after which the sample may be bleached and stained with the next biomarker and another set of images acquired. Multiple microscopes may be set up to process tissue samples in such a multiplexing workflow. However, in a conventional multiplexing workflow, all rounds of imaging for a given tissue sample are performed on a single microscope to eliminate variability that might otherwise be attributed to the differences in optical, geometric, and/or mechanical properties that may exist between different microscopes. By way of example, depending on the age and/or utilization of the lamps associated with each microscope in a fleet of microscopes, the brightness associated with each microscope may differ, leading to a lack of uniformity in terms of the illumination provided by each microscope.
With this in mind, one problem arising from such an approach for a given lab is how to load-balance a fleet of microscopes when, for example, one microscope is down for repair or is backed up with many tissue samples waiting for additional rounds of imaging while other microscopes sit idle. Among the problems that may exacerbate management of multiple microscopes in such an arrangement is the need to calibrate (e.g., optically, geometrically, mechanically, and so forth) all microscopes within a given lab relative to one another so that the layered images that are produced within a given lab are of high quality and are consistent over time, regardless of the microscope employed. Further, the calibration process should be as fast as possible to minimize downtime of the microscopes and to maximize the throughput (the number of tissue slides imaged per day) in the lab.